Bacteriocins are antimicrobial proteins or peptides that can be produced by certain bacteria, which can kill or inhibit the growth of closely related bacteria. The bacteriocins produced by lactic acid bacteria are of particular importance since they have great potential for the preservation of food and for the control of foodborne pathogens. (Wessels et al. 1998.)
The most well known bacteriocin is nisin, which is the only bacteriocin currently authorised as a food additive. Nisin is produced by fermentation of the dairy starter culture bacterium Lactococcus lactis subsp. lactis, and is sold as the commercial extract NISAPLIN® Natural Antimicrobial (Danisco). Nisin has an unusually broad antimicrobial spectrum for a bacteriocin, being active against most Gram-positive bacteria (e.g. species of Bacillus, Clostridium, Listeria, lactic acid bacteria). It is not normally effective against Gram-negative bacteria, yeasts or moulds. Nisin is allowed as a food preservative worldwide but its levels of use and approved food applications are strictly regulated, varying from country to country.
Other bacteriocins have since been discovered with potential as food preservatives, e.g. pediocin, lacticin, sakacin, lactococcin, enterococin, plantaricin, leucocin. These are also active, although usually with a more narrow spectrum, against Gram-positive bacteria. Their food use is at present restricted to production of the bacteriocin in situ, i.e. by growth of the producer organism within the food.
Antioxidants are widely used in food products susceptible to oxidative degeneration. An antioxidant is defined by the Food and Drug Administration (21CFR 170.3) as “a substance used to preserve food by retarding deterioration, rancidity, or discoloration due to oxidation”. Spices or plant extracts can be used in food as antioxidants and to impart flavour. One advantage of such extracts is that they are perceived as natural ingredients when compared to chemical antioxidants such as butyl hydroxyanisol (BHA) and butylated hydoxytoluene (BHT). Plants of the family Labiatae contain several well known herbs. Extracts from these plants have been shown to have antioxidant and, in some cases, antimicrobial activity (Nychas & Skandamis, 2003; Smid and Gorris, 1999; Loliger, 1989). Such extracts may be essential oils and oleoresins (extracts with essential oil content used in flavours and fragrances) or “deodorised’”, extracts that have a high phenolic diterpene content and low level of flavour-inducing compounds.
Essential oils are extracted by simple steam distillation of the plant material. The most effective antioxidant compounds in rosemary and sage are reported to be carnosic acid, camosol and rosmarinic acid (Cuvelier et al. 1996). Camosic acid, a phenolic diterpene (C20H28O4), occurs naturally in leaves of plants of the Labiatae family, particularly rosemary and sage, but also thyme and marjoram. Dried leaves of rosemary or sage contain 1.5-2.5% camosic acid and 0.3-0.4% camosol (U.S. Pat. No. 6,231,896). Camosol is an oxidative artefact of carnosic acid (Wenkert et al. J. Org. Chem 30:2931, 1965). The oxidation takes place in the presence of harvesting in the leaves left to dry in the air and if the leaves are subjected to extraction with solvents. Rosmanol may also be a product of the oxidation of camosic acid.
The use of extracts of plant material for inhibiting the growth of micro-organisms has been taught in the art. Examples of such teachings include: WO 02/069741 teaches Labiatae herb extracts and hop extracts for extending the colour lie and inhibiting the growth of micro-organisms in fresh meat, fish and poultry. Periago et al. 2001. Food Science & Technology International. 7: 487-492 relates to the use of Carvacrol and thymol at 0.3 mmol/liter in combination with nisin. It is taught that synergy is observed. JP 2001172159 relates to cosmetics comprising a range of components including antimicrobial agent and Labiatae solvent extract. WO 98/56395 teaches a mix of tea-tree oil and thyme Essential oil. GB 2275 194 A discusses plant extract disinfectant. U.S. Pat. No. 6,083,921 discusses a combination of plant extracts including one from Labiatae: Scutellaria, preferably root (Radix scutellariae). U.S. Pat. No. 5,472,684 teaches an oral composition for plaque and gingivitis containing thymol and eugenol
Food safety and prevention of food spoilage is an ever present concern worldwide, particularly with the increasing trend for convenience foods such as ready to eat meals, soups, sauces or snacks. Spoilage of food is a major economic problem for the food manufacturer. Food manufacturers need to protect the health and safety of the public by delivering products that are safe to eat. Such food must have a guaranteed shelf life, either at chilled or ambient temperature storage. Consumers prefer good tasting food of high quality—this is difficult to achieve with chemical preservatives, harsh heating regimes and other processing measures. Food safety and protection is best achieved with a multiple preservation system using a combined approach of milder processing and natural preservatives. Foodborne micro-organisms are also less able to adapt and grow in food preserved with different preservative measures.
There is much concern about food safety and the growth of food pathogens such as Listeria monocytogenes. This particular pathogen can grow at low temperatures, which are often used as an additional preservative measure. Foodborne pathogens can sometimes adapt to different preservatives and storage conditions, thus a combination of preservative measures can be more successful than individual measures.
There is an increasing need to develop economical, natural and effective food preservative systems to meet the public demand for convenient, natural, safe, healthy, good quality food products with guaranteed shelf life. Bacteriocins such as nisin can be used as preservatives in food to help meet this need. Nisin is a proven safe, natural preservative with GRAS status. Other bacteriocins can be used for preservation if produced in situ, by growth of the bacteriocin producer organism in the food.
In some cases the bacteriocin levels required to ensure preservation or food safety may prove uneconomical, or are below effective levels due to regulatory and legislation constraints. When bacteriocins are produced in situ, the resulting bacteriocin levels may not be high enough to achieve the required preservative effect.
The present invention alleviates the problems of the prior art.
In one aspect the present invention provides a composition comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
In one aspect the present invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
In one aspect the present invention provides use of (a) an antimicrobial material; and b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material; wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
In one aspect the present invention provides kit for preparing a composition as defined herein, the kit comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition, in separate packages or containers; optionally with instructions for admixture and/or contacting and/or use.
Aspects of the invention are defined in the appended claims.
Of the Labiatae plant family, rosemary and sage have antioxidant activity in foods that is mainly related to phenolic diterpenes such as camosic acid and camosol, as well as other phenolic compounds, including phenolic triterpenes such as betulinic acid, oleanolic acid and ursolic acid; and rosmarinic acid. Antimicrobial activity has been attributed to some of these compounds, all of which can be obtained by selective extraction from the plants. The phenolic diterpenes, phenolic triterpenes and rosmarinic acid are distinct from the essential oils and oleoresins that are often used in flavours and fragrances. The high flavour and odour levels of essential oils is not conducive to their use in food. One skilled in the art would expect a combination of an antimicrobial material and an extract from the Labiatae plant family to provide a simple additive bactericidal or bacteriostatic effect. However, in vitro studies described herein have demonstrated synergistic enhancement of bacteriocin activity by a deodorised extract of Rosmarinus officinalis. This enhanced activity was also observed in a food model, increasing bacteriocin (for example nisin) kill and growth control of Gram-positive bacteria. Enhanced bacteriocin activity was also observed with rosemary extracts specifically prepared to contain high levels of the phenolic diterpenes camosol and camosic acid, indicating these compounds play an important role in the synergy. Enhanced bacteriocin activity was also observed with rosmarinic acid.
The present invention provides a synergistic combination of components for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, such as foodstuff. This combination of components allows lower levels of the antimicrobial material to be used to provide effective action and prevent the development of tolerance to the antimicrobial material. This is particularly important in food applications where reduction of dosage and/or avoidance of development of tolerance is desired for commercial and regulatory reasons.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
Preferred Aspects
Antimicrobial Material
In one preferred aspect the antimicrobial material is a bacteriocin.
The antimicrobial material, such as a bacteriocin, may typically be selected from materials (bacteriocins) that can be used as preservatives in food
Preferably the antimicrobial material is selected from lanthionine containing bacteriocins, Lactococcus-derived bacteriocins, Streptococcus-derived bacteriocins, Pediococcus-derived bacteriocins, Lactobacillus-derived bacteriocins, Camobacterium-derived bacteriocins, Leuconostoc-derived bacteriocins, Enterococcus-derived bacteriocins and mixtures thereof
Preferably the antimicrobial material is at least nisin.
Preferably the antimicrobial material consists of nisin.
Nisin is a lanthionine-containing bacteriocin (U.S. Pat. No. 5,691,301) derived from Lactococcus lactis subsp. lactis (formerly known as Streptococcus-lactis) (U.S. Pat. No. 5,573,801). In a preferred aspect of the present invention the bacteriocin used in the present invention is at least nisin.
As discussed in U.S. Pat. No. 5,573,801 nisin is a polypeptide bacteriocin produced by the lactic acid bacteria, Lactococcus lactis subsp. lactis (formerly known as Streptococcus lactis Group N).
Nisin is reportedly a collective name representing several closely related substances which have been designated nisin compounds A, B, C, D and E (De Vuyst, L. and Vandamme, E. J. 1994. Nisin, a lantibiotic produced by Lactococcus lactis subsp. lactis: properties, biosynthesis, fermentation and applications. In: Bacteriocins of lactic acid bacteria. Microbiology, Genetics and Applications. Eds.: De Vuyst and Vandamme. Blackie Academic and Professional, London). The structure and properties of nisin are also discussed in the article by E. Lipinska, entitled “Nisin and Its Applications”, The 25th Proceedings of the Easter School in Agriculture Science at the University of Nottingham, 1976, pp. 103-130 (1977), which article is hereby incorporated by reference. In 1969 the FAO/WHO Joint Expert Committee on Food Additives set specifications for the purity and identity of nisin (FAO/WHO Joint Expert Committee on Food Additives. 1969. Specifications for identity and purity of some antibiotics. 12th Report. WHO Technical Report Series No. 430). This committee recognised nisin as a safe and legal preservative based on extensive toxicological testing. Nisin has the food additive number E234 and is classed as GRAS (Generally Recognised As Safe) (Food and Drug Administration. 1988. Nisin preparation: Affirmation of GRAS status as a direct human ingredient. Federal Regulations 53: 11247). The international activity unit (IU hereinafter) was defined as 0.001 mg of an international nisin reference preparation. NISAPLIN® Natural Antimicrobial is the brand name for a nisin concentrate containing 1 million IU per g, which is commercially available from Danisco.
Nisin is an acknowledged and accepted food preservative with a long history of safe, effective food use. There have been several reviews of nisin, e.g. Hurst 1981; 1983; Delves-Broughton, 1990; De Vuyst and Vandamme, 1994; Thomas et al. 2000; Thomas & Delves-Broughton, 2001). Nisin was discovered over 50 years ago and the first commercial preparation, made in 1953, was NISAPLIN®. Nisin has several characteristics that make it particularly suitable as a food preservative. It has undergone extensive toxicological testing to demonstrate its safety. It is heat-stable, acid-stable and effective against a broad spectrum of Gram-positive bacteria. It is not normally effective against Gram-negative bacteria, yeasts or moulds but activity against Gram-negative bacteria and yeasts has been reported in the presence of chelating agents (PCT/US 8902625. WO 89/12399). Nisin is an effective preservative in pasteurised and heat-treated foods (e.g. processed cheese, cheese, pasteurised milks, dairy desserts, cream, mascarpone and other dairy products, puddings such as semolina, tapioca etc., pasteurised liquid egg, pasteurised potato products, soy products, crumpets, pikelets, flapjacks, processed meat products, beverages, soups, sauces, ready to eat meals, canned foods, vegetable drinks) and low acid foods such as salad dressings, sauces, mayonnaise, beer, wine and other beverages.
Although some loss of activity may be expected when used with processed foods, this may be ameliorated e.g. by increasing the amount of nisin applied. Effective levels of nisin to preserve foodstuffs reportedly range from 25-500 IU/g or more. Other effective levels would be appreciated by one skilled in the art. For example levels of 50-400 IU/g may be utilised.
Since the discovery of the first bacteriocin, nisin, many other bacteriocins have now been found (Hoover, 1993; Ray & Daeschel, 1994; Axelsen, 1998; Naidu, 2000; Ray at al. 2001; Ray & Miller, 2003). The bacteriocin pediocin, produced by Pediococcus pentosaceus, P. acidilactici, or Lactobacillus plantarum, may be used in the present invention. Like nisin, different structures of pediocin have been described. At present pediocin and other bacteriocins are not allowed as food additives but their antibacterial activity can be achieved by production of the bacteriocin in situ, as a consequence of the growth of the producer organism in the food. This is the purpose of commercial protective cultures such as HOLDBAC™ Listeria (Danisco). Pediocin has a more narrow antimicrobial spectrum compared to nisin, but there is much interest in its food safety ability to kill, prevent or control the growth of the food pathogen Listeria monocytogenes (Ray & Miller, 2000). Other bacteriocins may be used in the present invention, including those named generally as divercin, leucctcin, mesentericin, sakacin, curvacin, bavaricin, acidocin, bifidocin, camobacteriocin, pisicocin, piscicolin, mundticin, enterocin, thermophilin, lacticin, plantaricin, lactococcin, divercin, diplococcin, mesenterocin, leuconosin, camosin, acidophilin, lactacin, brevicin, lactocin, helevticin, reutericin, propionicin.
Extract
As discussed herein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.05 wt. % based on the composition, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect, the composition comprises carvacrol in an amount of less than 0.05 wt. % based on the composition, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises carvone in an amount of less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect the composition comprises carvone in an amount of less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises thymol in an amount of less than 15 wt. % based on the composition, preferably less than 10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect the composition comprises thymol in an amount of less than 15 wt. % based on the composition preferably less than 10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises eugenol in an amount of less than 15 wt. % based on the composition, preferably less than 10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect the composition comprises eugenol in an amount of less than 15 wt. % based on the composition preferably less than 10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and each of carvone and thymol in amounts of less than 15 wt. % based on the composition (preferably less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition).
In one preferred aspect, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and each of carvone and thymol in amounts of less than 15 wt. % based on the composition (preferably less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition).
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and each of carvone, thymol and eugenol in amounts of less than 15 wt. % based on the composition (preferably less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition).
In one preferred aspect, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and each of carvone, thymol and eugenol in amounts of less than 15 wt. % based on the composition (preferably less than 10 wt. % based on the composition, preferably less than 7 wt. %, preferably less than 5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt. %, preferably less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the composition).
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises each of carvacrol and carvone in an amount of less than 1 wt. % based on the extract. Preferably when the antimicrobial material consists of nisin the composition comprises each of carvacrol and carvone in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the extract.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises each of carvacrol, carvone and thymol in amounts of less than 1 wt. % based on the extract. Preferably when the antimicrobial material consists of nisin the composition comprises each of carvacrol, carvone and thymol in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the extract.
In one preferred aspect when the antimicrobial material consists of nisin, the composition comprises each of carvacrol, carvone, thymol and eugenol in amounts of less than 1 wt. % based on the extract. Preferably when the antimicrobial material consists of nisin the composition comprises each of carvacrol, carvone, thymol and eugenol in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01%4.%, preferably less than 0.004 wt. %, based on the extract.
In one preferred aspect the composition comprises each of carvacrol and carvone in an amount of less than 1 wt. % based on the extract. Preferably the composition comprises each of carvacrol and carvone in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the extract.
In one preferred aspect the composition comprises each of carvacrol, carvone and thymol in amounts of less than 1 wt. % based on the extract. Preferably the composition comprises each of carvacrol, carvone and thymol in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the extract.
In one preferred aspect the composition comprises each of carvacrol, carvone, thymol and eugenol in amounts of less than 1 wt. % based on the extract. Preferably the composition comprises each of carvacrol, carvone, thymol and eugenol in an amount of less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less than 0.004 wt. %, based on the extract.
The extract used in the present invention is obtained from or is obtainable from a plant of the Labiatae family.
In one aspect the extract used in the present invention is obtained from a plant of the Labiatae family.
It will be appreciated by one skilled in the art that by the term “extract” or “extracts” it is meant any constituent of the plant which may be isolated from the whole plant.
In one aspect the extract used in the present invention is obtainable from a plant of the Labiatae family. It will be appreciated by one skilled in the art that an extract obtainable from a plant may be obtained from a plant or may be isolated from the plant, identified and then obtained from an alternative source, for example by chemical synthesis or enzymatic production. For example the extract may be produced by a eukaryotic or prokaryotic fermentation, by a process of genetic manipulation. The present applicant have recognised that products present in a plant of the Labiatae family may synergistically increase the activity of a an antimicrobial material, preferably a bacteriocin. These products may be obtained from any source and will fall within the scope of the present invention.
The invention comprises use of a combination of a bacteriocin such as nisin and a of the Labiatae plant family, such as rosemary (Rosmarinus officinalis) or sage (Salvia officinalis) that together give enhanced control of Gram-positive bacteria in a food system. The extracts responsible for synergy in the present invention preferably refer to extracts of the plant family Labiatae that have been selectively extracted (“deodorised extracts”) to increase their phenolic diterpene content (such as carnosol and camosic acid), phenolic triterpene content (such as ursolic acid, betulinic acid and oleanolic acid) or rosmarinic acid content. These deodorised extracts can be distinguished by their high phenolic diterpene content (for example greater than 3.5 wt. %) and their low level (less than 1 wt. %) of flavour-inducing compounds from plant essential oils and oleoresins that are used as flavours or fragrances. Essential oils are typically extracted by simple steam distillation of the plant material.
Essential oils comprise the various essential oils in plants having the odour or the flavour of the plant from which they were extracted. The essential oils are typically terpenoids often comprising monoterpenes. For example an antioxidant type of rosemary extract, which could be described as selectively extracted or deodorised, contains >3.5% phenolic diterpenes but less than 1 wt. % essential oils. A non-selective, flavouring extract contains 10-30 wt. % essential oils and a phenolic diterpene content of 2→3.5 wt. %.
An essential oil is commonly described as the volatile ethereal fraction obtained from a plant or plant part by a physical separation process such as distillation or chromatographic separation. Essential oils have also been described as a group of odorous principles, soluble in alcohol and to a limited extent in water, consisting of a mixtures of esters, aldehydes, ketones and terpenes. Essential oils are typically obtained by distilling plants with water, the oil that separates from distillate usually has highly characteristic odors identified with the plant origin. The resulting mixture of organic compounds was thought, in the days of alchemists, to be the essence of the plant, hence the term “essential oil”.
In one preferred aspect the extract is a deodorised extract. Preferably the (deodorised) extract contains from 1.0 to 70 wt. % phenolic diterpenes, preferably 3.5 to 70 wt. % phenolic diterpenes and less than 1 wt. % essential oil.
In one preferred aspect the extract is selected from phenolic diterpenes, phenolic triterpenes and rosmarinic acid.
In one preferred aspect the extract is or comprises a phenolic diterpene. Preferably the phenolic diterpene is selected from carnosic acid, carnosol and methylcamosic acid. Preferably the phenolic diterpene is selected from camosic acid and carnosol.
In one preferred aspect the combined amount of phenolic diterpenes, and phenolic triterpenes and rosmarinic acid, based on the extract, is greater than 1.0 wt. %. In one preferred aspect the combined amount of phenolic diterpenes, and phenolic triterpenes and rosmarinic acid, based on the composition, is greater than 1.0 wt. %.
In one preferred aspect the combined amount of phenolic diterpenes, and phenolic triterpenes and rosmarinic acid, based on the extract, is greater than 3.5 wt. %. In one preferred aspect the combined amount of phenolic diterpenes, and phenolic triterpenes and rosmarinic acid, based on the composition, is greater than 3.5 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the extract, is greater than 1.0 wt. %, for example greater than 5.0 wt. %, greater than 10.0 wt. %, greater than 20.0 wt. %, or greater than 25.0 wt. %. In one preferred aspect the amount of phenolic diterpenes, based on the composition, is greater than 1.0 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the extract, is greater than 3.5 wt. %. In one preferred aspect the amount of phenolic diterpenes, based on the composition, is greater than 3.5 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the composition, is greater than 1.0 wt. %, preferably greater than 2.0 wt. %, preferably greater than 3.0 wt. %, preferably greater than 3.5 wt. %, preferably greater than 5.0 wt. %, preferably greater than 10.0 wt. %, preferably greater than 20.0 wt. %, preferably greater than 40.0 wt. %, preferably greater than 50.0 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the composition, is from 2.0 to 2.5 wt. %, such as 2.3 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the composition, is from 4.0 to 4.5 wt. %, such as 4.2 wt. %.
In one preferred aspect the amount of phenolic diterpenes, based on the extract, is greater than 1.0 wt. %, preferably greater than 2.0 wt. %, preferably greater than 3.0 wt. %, preferably greater than 3.5 wt. %, preferably greater than 5.0 wt. %, preferably greater than 10.0 wt. %, preferably greater than 20.0 wt. %, preferably greater than 40.0 wt. %, preferably greater than 50.0 wt. %.
In one highly preferred aspect the extract contains one or more phenolic triterpenes. Preferably the phenolic triterpenes are selected from betulinic acid, oleanolic acid, and ursolic acid.
In one preferred aspect is or comprises a phenolic triterpene. Preferably the phenolic triterpene is selected from betulinic acid, oleanolic acid, and ursolic acid.
In one highly preferred aspect the amount of phenolic triterpenes, based on the extract, is greater than 3.5 wt. %. In one highly preferred aspect the amount of phenolic triterpenes, based on the composition, is greater than 3.5 wt. %.
In one preferred aspect the extract is or comprises rosmarinic acid.
In one preferred aspect the amount of rosmarinic acid, based on the extract, is greater than 3.5 wt. %. In one preferred aspect the amount of rosmarinic acid, based on the composition, is greater than 3.5 wt. %.
In one preferred aspect the extract contains flavour-inducing compounds and/or essential oils in an amount of less than 1 wt. % based on the extract. In one preferred aspect the extract contains flavour-inducing compounds and/or essential oils in an amount of less than 1 wt. % based on the composition.
Typically flavour-inducing compounds and/or essential oils are camphor, verbenone, borneol and alfa-terpineol.
In one preferred aspect the combined amount of camphor present in the extract is less than 1 wt. % (preferably less than 0.2 wt. %, more preferably less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on the extract.
In one preferred aspect the combined amount of verbenone present in the extract is less than 1 wt. % (preferably less than 0.2 wt. %, more preferably less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on the extract.
In one preferred aspect the combined amount of borneol present in the extract is less than 1 wt. % (preferably less than 0.2 wt. %, more preferably less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on the extract.
In one preferred aspect the combined amount of alfa-terpineol present in the extract is less than 1 wt. % (preferably less than 0.2 wt. %, more preferably less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on the extract.
In one preferred aspect the combined amount of camphor, verbenone, borneol and alfa-terpineol present in the extract is less than 1 wt. % (preferably less than 0.2 wt. %, more preferably less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on the extract.
In one preferred aspect the extract contain less than 1 wt. % of plant essential oils and/or oleoresins based on the extract. In one preferred aspect the extract contain less than 1 wt. % of plant essential oils and/or oleoresins based on the composition.
In one preferred aspect the extract contains essential oils in an amount of less than 1 wt. % based on the extract. In one preferred aspect the extract contains essential oils in an amount of less than 1 wt. % based on the composition.
In one preferred aspect the plant of the Labiatae family is selected from rosemary, sage, oregano, marjoram, mint, balm, savoury and thyme. In one preferred aspect the plant of the Labiatae family is selected from rosemary, sage, oregano, marjoram, mint, balm, and savoury. It will be understood that these name cover all species and varieties of plants known by these names.
In one preferred aspect the plant of the Labiatae family is selected from rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L) oregano (Origanum vulgare L.), marjoram (Origanum marjorana L.), mint (Mentha spp.), balm (Melissa officinalis L.), savoury (Satureia hortensis), thyme (Thymus vulgaris L.).
In one preferred aspect the plant of the Labiatae family is selected from rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), oregano (Origanum vulgare L.), marjoram (Origanum marjorana L.), mint (Mentha spp.), balm (Melissa officinalis L.), and savoury (Satureia hortensis).
In one preferred aspect the plant of the Labiatae family is selected from rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), marjoram (Origanum marjorana L.), mint (Mentha spp.), balm (Melissa officinalis L), and savoury (Satureia hortensis).
In one preferred aspect the plant of the Labiatae family is rosemary.
In a further preferred aspect the phenolic diterpenes, phenolic triterpenes and rosmarinic acid are obtained by chemical synthesis.
Thus in highly preferred aspects the present invention provides                a composition comprising (a) an antimicrobial material and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid (preferably camosic acid and camosol).        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid (preferably camosic acid and carnosol).        use of (a) an antimicrobial material and (b) a compound selected from camosic acid, carnosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid (preferably camosic acid and carnosol), for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material.Microorganism        
As discussed herein the present invention may prevent and/or inhibit the growth of, and/or kill a micro-organism in a material. This may be slowing or arresting a micro-organism, such a bacteria, or by killing the micro-organism present on contact with the present composition.
In one aspect the antimicrobial material and/or the extract are present in an amount to provide a microbicidal or microbiostatic effect.
In one aspect the bacteriocin and the extract are present in an amount to provide a microbicidal or microbiostatic effect.
In one aspect the bacteriocin and the extract are present in an amount to provide a microbicidal or microbiostatic synergistic effect.
In one aspect the bacteriocin and the extract are present in an amount to provide a microbicidal synergistic effect.
In a highly preferred aspect the microbicidal or microbiostatic effect is a bactericidal or bacteriostatic effect.
It is advantageous for the bactericidal or bacteriostatic effect to be in respect of Gram-positive bacteria and Gram-negative bacteria. Preferably the bactericidal or bacteriostatic effect is in respect of Gram-positive bacteria.
In a preferred aspect the bactericidal or bacteriostatic effect is in respect of an organism selected from Gram-positive bacteria associated with food spoilage or foodborne disease including Bacillus species, Bacillus subtilis, Bacillus cereus, Listeria species, Listeria monocytogenes, lactic acid bacteria, lactic acid spoilage bacteria, Lactobacillus species, Staphylococcus aureus, Clostridium species, C. sporogenes, C. tyrobutyricum. 
In a preferred aspect the bactericidal or bacteriostatic effect of the invention in combination with a chelating agent is in respect of an organism selected from other micro-organisms associated with food spoilage or foodborne disease, including yeasts, moulds and Gram-negative bacteria including Escherichia coli, Salmonella species, and Pseudomonas species.
In a preferred aspect the bactericidal or bacteriostatic effect is in respect of an organism selected from Bacillus cereus 204, B. cereus Campden, B. cereus NCTC2599, B. subtilis Campden, Clostridium sporogenes strain Campden, Clostridium sporogenes strain 1.221, Clostridium sporogenes NCIMB1793, Listeria monocytogenes 272, L. monocytogenes NCTC12426, L. monocytogenes S23, Lactobacillus sake 272, Escherichia coli S15, E. coli CRA109, Salmonella Typhimurium S29, Pseudomonas fluorescens 3756,
In a preferred aspect the bactericidal or bacteriostatic effect is in respect of Listeria monocytogenes. 
Foodstuff
The composition, process and use of the present invention may prevent and/or inhibit the growth of, and/or kill a micro-organism in any material. However, in view of the problems associated with spoilage and contamination of foodstuffs and in view of the particular effectiveness of the present invention in foodstuffs, preferably the composition is a foodstuff or may be added to a foodstuff. It will be appreciated by one skilled in the art that when the present composition is a foodstuff the essential components of (a) an antimicrobial material and (b) a extract obtained from or obtainable from a plant of the Labiatae family are already present in the foodstuff. They may have been provided by one or more means. For example they may have been added in the form of a composition containing the bacteriocin and the extract. The two components (the bacteriocin and the afore mentioned extract) may have been added to the foodstuff sequentially. In one further aspect one or more of the components may have be formed in situ in the foodstuff. For example the bacteriocin may be formed in situ in the foodstuff by fermentation of the dairy starter culture bacterium Lactococcus lactis subsp. lactis. 
In one aspect the composition of the present invention is a protectant composition suitable for addition to a foodstuff.
Many foodstuffs may be protected by the present invention. Typical foodstuffs are raw meat, cooked meat, raw poultry products, cooked poultry products, raw seafood products, cooked seafood products, ready to eat meals, pasta sauces, pasteurised soups, mayonnaise, salad dressings, oil-in-water emulsions, margarines, low fat spreads, water-in-oil emulsions, dairy products, cheese spreads, processed cheese, dairy desserts, flavoured milks, cream, fermented milk products, cheese, butter, condensed milk products, ice cream mixes, soya products, pasteurised liquid egg, bakery products, confectionery products, fruit products, and foods with fat-based or water-containing fillings.
Additional Components
The composition of the present invention or the composition for use in the present invention may contain one or more additional components. However, in some aspects the protectant composition of the present invention (suitable for addition to a foodstuff) contains no additional components or contains no additional components that materially affect the properties of the composition. In these aspects the present invention provides                a composition consisting essentially of (a) a bacteriocin and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different        a composition consisting of (a) a bacteriocin and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different        a composition consisting essentially of (a) a bacteriocin and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different, wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a composition consisting of (a) a bacteriocin and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different, wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        
In one preferred aspect the composition further comprises an emulsifier. Preferably the emulsifier is selected from polyoxy-ethylene sorbitan esters (E432-E436) otherwise known as polysorbates (e.g. Tween 80, Tween 20), monoglycerides, diglycerides, acetic acid esters of mono-diglycerides, tartaric acid esters of mono-diglycerides and citric acid esters of mono-diglycerides.
In one preferred aspect the composition further comprises a chelator. Preferably the chelator is selected from EDTA, citric acid, monophosphates, diphosphates, triphosphates and polyphosphates.
Further suitable chelator are taught in U.S. Pat. No. 5,573,801 and include carboxylic acids, polycarboxylic acids, amino acids and phosphates. In particular, the following compounds and their salts may be useful:
Acetic acid, Adenine, Adipic acid, ADP, Alanine, B-Alanine, Albumin, Arginine, Ascorbic acid, Asparagine, Aspartic acid, ATP, Benzoic acid, n-Butyric acid, Casein, Citraconic acid, Citric acid, Cysteine, Dehydracetic acid, Desferri-ferrichrysin, Desferri-ferrichrome, Desferri-ferrioxamin E, 3,4-Dihydroxybenzoic acid, Diethylenetriaminepentaacetic acid (DTPA), Dimethylglyoxime, O,O-Dimethylpurpurogallin, EDTA, Formic acid, Fumaric acid, Globulin, Gluconic acid, Glutamic acid, Glutaric acid, Glycine, Glycolic acid, Glycylglycine, Glycylsarcosine, Guanosine, Histamine, Histidine, 3-Hydroxyflavone, Inosine, Inosine triphosphate, Iron-free ferrichrome, Isovaleric acid, Itaconic acid, Kojic acid, Lactic acid, Leucine, Lysine, Maleic acid, Malic acid, Methionine, Methylsalicylate, Nitrilotriacetic acid (NTA), Omithine, Orthophosphate, Oxalic acid, Oxystearin, B-Phenylalanine, Phosphoric acid, Phytate, Pimelic acid, Pivalic acid, Polyphosphate, Proline, Propionic acid, Purine, Pyrophosphate, Pyruvic acid, Riboflavin, Salicylaldehyde, Salicyclic acid, Sarcosine, Serine, Sorbitol, Succinic acid, Tartaric acid, Tetrametaphosphate, Thiosulfate, Threonine, Trimetaphosphate, Triphosphate, Tryptophan, Uridine diphosphate, Uridine triphosphate, n-Valeric acid, Valine, andXanthosine
Many of the above sequestering agents are useful in food processing in their salt forms, which are commonly alkali metal or alkaline earth salts such as sodium, potassium or calcium or quaternary ammonium salts. Sequestering compounds with multiple valencies may be beneficially utilised to adjust pH or selectively introduce or abstract metal ions e.g. in a food system coating. Additional information chelators is disclosed in T. E. Furia (Ed.), CRC Handbook of Food Additives, 2nd Ed., pp. 271-294 (1972, Chemical Rubber Co.), and M. S. Peterson and A. M. Johnson (Eds.), Encyclopaedia of Food Science, pp. 694-699 (1978, AVI Publishing Company, Inc.) which articles are both hereby incorporated by reference.
The terms “chelator” is defined as organic or inorganic compounds capable of forming co-ordination complexes with metals. Also, as the term “chelator” is used herein, it includes molecular encapsulating compounds such as cyclodextrin. The chelator may be inorganic or organic, but preferably is organic.
Preferred chelator are non-toxic to mammals and include aminopolycarboxylic acids and their salts such as ethylenediaminetetraacetic acid (EDTA) or its salts (particularly its di- and tri-sodium salts), and hydrocarboxylic acids and their salts such as citric acid. However, non-citric acid and non-citrate hydrocarboxylic acid chelators are also believed useful in the present invention such as acetic acid, formic acid, lactic acid, tartaric acid and their salts.
As noted above, the term “chelator” is defined and used herein as a synonym for sequestering agent and is also defined as including molecular encapsulating compounds such as cyclodextrin. Cyclodextrins are cyclic carbohydrate molecules having six, seven, or eight glucose monomers arranged in a donut shaped ring, which are denoted alpha, beta or gamma cyclodextrin, respectively. As used herein, cyclodextrin refers to both unmodified and modified cyclodextrin monomers and polymers. Cyclodextrin molecular encapsulators are commercially available from American Maize-Products of Hammond, Ind. Cyclodextrin are further described in Chapter 11 entitled, “Industrial Applications of Cyclodextrin”, by J. Szejtli, page 331-390 of Inclusion Compounds, Vol. III (Academic Press, 1984) which chapter is hereby incorporated by reference.
Preferably the chelator enhances the antimicrobial activity and/or antimicrobial spectrum of the bacteriocin. More preferably the chelator enhances the antimicrobial activity and/or antimicrobial spectrum of the bacteriocin in respect of Gram-negative bacteria and other micro-organisms.
In one preferred aspect the composition further comprises a lytic enzyme. Preferably the lytic enzyme is a lysozyme.
Process
As discussed herein in one aspect the present invention provides process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
In one aspect the bacteriocin and the extract are added to the material together.
In one aspect the bacteriocin and the extract are added to the material sequentially.
Thus the present invention provides in one aspect a preservative/protectant composition which may be added to a range of materials such as food systems and in another aspect a combination of two separate products which may added sequentially to materials such as food products.
In one aspect the extract is added to the material.
In one aspect the bacteriocin is added to the material.
In one aspect the extract is formed in situ in the material.
In one aspect the bacteriocin is formed in situ in the material. Preferably when the bacteriocin is nisin, the bacteriocin may be formed in situ in the foodstuff by fermentation of the dairy starter culture bacterium Lactococcus lactis subsp. lactis. 
Highly Preferred Aspects
As discussed herein in one aspect the present invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and can/one in an amount of less than 15 wt. % based on the composition.
In one aspect the present invention provides use of (a) an antimicrobial material; and b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material; wherein (a) and (b) are different; and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.
Some highly preferred aspects of the present invention are set out below                a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from a plant of the Labiatae family, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition        a composition comprising (a) a bacteriocin, and (b) an extract obtained from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, wherein (a) and (b) are different        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, wherein (a) and (b) are different        use of (a) a bacteriocin, and (b) an extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein (a) and (b) are different        a composition comprising (a) a bacteriocin, and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid.        use of (a) a bacteriocin, and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material.        a composition comprising (a) a bacteriocin, and (b) camosic acid.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, and (b) camosic acid.        use of (a) a bacteriocin, and (b) camosic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, thyme, mint, balm, savoury, sage and oregano, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; wherein the bacteriocin is nisin; and (b) a selectively extracted extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a selectively extracted extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, carnosol, methylcamosic acid, betulinic acid, oloanolic acid, ursolic acid and rosmarinic acid, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oloanolic acid, ursolic acid and rosmarinic acid, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, carnosol, methylcarnosic acid, betulinic acid, oloanolic acid, ursolic acid and rosmarinic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) camosic acid.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) camosic acid.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) camosic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, wherein the bacteriocin and the extract are present in an amount to provide a bactericidal or bacteriostatic synergistic effect, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, wherein the bacteriocin and the extract are present in an amount to provide a bactericidal or bacteriostatic synergistic effect wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family selected from rosemary, sage, thyme, mint, balm, savoury and oregano, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the bacteriocin and the extract are present in an amount to provide a bactericidal or bacteriostatic synergistic effect, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, camosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, carnosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from carnosic acid, carnosol, methylcamosic acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from camosic acid, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) a compound selected from carnosic acid, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the bacteriocin and the compound are present in an amount to provide a bactericidal or bacteriostatic synergistic effect        a composition comprising (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from a plant of the Labiatae family, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, wherein the composition comprises carvacrol in an amount of less than 0.04 wt. % based on the composition and carvone in an amount of less than 0.04.% based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) a bacteriocin; wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, wherein the composition comprises carvacrol in an amount of less than 0.04 wt. % based on the composition and carvone in an amount of less than 0.04.% based on the composition.        use of (a) a bacteriocin, wherein the bacteriocin is nisin; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, wherein the composition comprises carvacrol in an amount of less than 0.04 wt. % based on the composition and carvone in an amount of less than 0.04.% based on the composition.        
Further broad aspects of the present invention are defined below:                a composition comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %.        use of (a) an antimicrobial material; and b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material; wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %.        a kit for preparing a composition as defined herein, the kit comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition.        a composition comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, the process comprising the step of contacting the material with (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        use of (a) an antimicrobial material; and b) an extract obtained from or obtainable from a plant of the Labiatae family, for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material; wherein (a) and (b) are different; wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.        a kit for preparing a composition as defined herein, the kit comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition, in separate packages or containers; optionally with instructions for admixture and/or contacting and/or use.        
The present invention will now be described in further detail in the following examples.